Compositions for improving the flavor of alcoholic beverage made from grape

ABSTRACT

It is intended to provide novel compositions usable in improving the flavor of an alcoholic beverage made from grapes typified by wine. Namely, a composition usable in improving the flavor of an alcoholic beverage made from grapes which contains the culture of a strain belonging to a genus  Aspergillus, Penicillium, Rhizopus, Rhizomucor, Talaromyces, Mortierella, Cryptococcus, Microbacterium, Corynebacterium  or  Actinoplanes  and being capable of producing diglycosidase.

TECHNICAL FIELD

This invention relates to compositions employed to improve the flavorsof alcoholic beverages made from grapes. The present inventionadditionally relates to methods of manufacturing grape-based alcoholicbeverages with such compositions and to grape-based alcoholic beveragesmanufactured with such compositions. Application of the presentinvention will provide grape-based alcoholic beverages with improvedflavors, such as wine with superior flavor.

BACKGROUND ART

Broad varieties of wine having distinctive flavors attributable to theirplaces of origin and the different manufacturing methods are indistribution today. The flavor of wine is primarily determined by thequality of its ingredient, grape.

Meanwhile, efforts are being made to increase the amounts of thecomponents responsible for the aroma of wine so as to provide wine thatis rich in aroma, i.e., wine with improved flavor. A known method ofimproving the flavor of wine involves addition of β-glucosidase in awine making process so as to increase the aroma of the wine through theoperation of this enzyme.

Monoterpenes, which are the components responsible for the aromacharacteristic of wine made from muscat grapes and German grapes, suchas Riesling and Kelner, exist in grapes mainly as glycosides ofdisaccharides (a-L-arabinofuranosyl-β-D-glucopyranoside,a-L-rhamnopyranosyl-β-D-glucopyranoside, andapiofuranosyl-β-D-glucopyranoside).

β-glucosidase exhibits a low glucose resistance. Accordingly, inwinemaking, β-glucosidase cannot be used immediately before thefermentation step, when the glucose concentration is high; rather, itshould be added in the advanced stage of fermentation when the glucoseconcentration has become lower in the crude wine if the enzyme is towork. Accordingly, if used at all, β-glucosidase has been added to crudewine after the fermentation is completed (for example, added in theracking step (the step of decanting supernatant)) This means that such amethod using β-glucosidase has constraints on the timing of initiatingthe process of enzymatic reaction, making the method less thanconvenient and flexible.

Today, consumers' preferences have been increasingly diversified, suchthat there is a greater demand for wine with novel flavors. In view ofsuch a demand, it has been desired to develop a method of making winethat augments the components responsible for the aroma of wine throughnovel mechanisms.

In view of the foregoing problems, an object of the present invention isto provide novel compositions that can be employed to improve theflavors of grape-based alcoholic beverages, most notably wine. Inaddition, other objects of the present invention include provision ofmethods of manufacturing grape-based alcoholic beverages with improvedflavors and provision of grape-based alcoholic beverages with improvedflavors. Moreover, other objects of the invention are to provide methodsof manufacturing such grape-based alcoholic beverages withoutcomplicating their manufacturing processes and to provide suchgrape-based alcoholic beverages without complicating their manufacturingprocesses.

DISCLOSURE OF INVENTION

In view of the foregoing objects, the inventors have selected wine, atypical grape-based alcoholic beverage, as a model to improve theflavors of such beverages. The inventors first paid attention to theaction of diglycosidases and eventually conceived the idea of usingcultures of microorganisms that produce diglycosidases as suchflavor-improving compositions. When added to the ingredients to operateduring the fermentation step of wine, these compositions efficientlybrought out the components responsible for aroma in the ingredients andthus produced wine with superior flavor. This led to the knowledge thatsuch compositions including diglycosidases are useful in improving theflavors of wine and other alcoholic beverages made from grapes.Furthermore, these compositions are capable of operation in thefermentation steps in which conventional β-glucosidases cannot operate,showing that these compositions improve the wine flavor through adifferent mechanism from the one through which β-glucosidases improvethe flavors of wine. It has been also learned that these compositionsoffers other advantages in winemaking processes. For example, thesecompositions can be removed together with the yeast following thefermentation step.

The present invention is based on these findings and provides thefollowing:

(1) A composition for improving the flavor of an alcoholic beverage madefrom grapes, the composition containing a culture of a strain belongingto Genus Aspergillus, Genus Penicillium, Genus Rhizopus, GenusRhizomucor, Genus Talaromyces, Genus Mortierella, Genus Cryptococcus,Genus Microbacterium, Genus Corynebacterium, or Genus Actinoplanes, andbeing capable of producing diglycosidase.

(2) A composition as set forth in Section (1), wherein the strainbelongs to Aspergillus niger, Aspergillus fumigatus, or Penicilliummulticolor.

(3) A composition as set forth in Section (1), wherein the strainbelongs to Aspergillus niger IFO4407, Aspergillus niger IAM2020,Aspergillus fumigatus IAM2046, or Penicillium multicolor IAM7153.

(4) A composition including an extracellular enzyme produced byPenicillium multicolor IAM7153.

(5) A composition as set forth in any one of Sections (1) to (4),wherein the diglycosidase activity is 0.0001 units/mg or more in dryweight.

(6) A method of manufacturing a grape-based alcoholic beverage,including an enzymatic process step of causing any one of theflavor-improving compositions set forth in Sections (1) to (5) tooperate.

(7) A method of manufacturing as set forth in Section (6), wherein theenzymatic process step includes addition of the flavor-improvingcomposition in part of the process of manufacturing the grape-basedalcoholic beverage.

(8) A method of manufacturing as set forth in Section (7), wherein thepart of the process of manufacturing includes one or more steps selectedfrom the group consisting of crushing and destemming, squeezing,fermentation, sediment removal, and aging.

(9) A method of manufacturing a grape-based alcoholic beverage,characterized by adding any one of the compositions set forth inSections (1) to (5) in the fermentation and/or aging step.

(10) A method of manufacturing a grape-based alcoholic beverage as setforth in any one of Sections (6) to (9), wherein the grape-basedalcoholic beverage is wine.

(11) A grape-based alcoholic beverage manufactured by any one of themethods set forth in Sections (6) to (10).

As used in this specification, the term “improving flavor” is intendedto include augmenting the flavor of an alcoholic beverage made fromgrapes, thus increasing the amounts of the components in the beverageresponsible for its aroma and enriching the flavor of the beverage. Forexample, the term includes augmentation of the fragrance and mellownessof such a beverage. As used in this specification, the term “improvingflavor” is also intended to include augmenting the amount of a specificcomponent responsible for the aroma of an alcoholic beverage and thusindirectly affecting how the other aromatic components are sensed,thereby affecting the overall aroma of the beverage as sensed by aperson.

Furthermore, as used in this specification, the term “standardmanufacturing process” is intended to mean a process of making analcoholic beverage from grapes that does not include the enzymaticprocess step according to the present invention.

Moreover, as used in this specification, the term “percent (%)” isintended to mean % (w/v).

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect, the present invention relates to compositions forimproving the flavor of an alcoholic beverage made from grapes. Theflavor-improving compositions of the present invention (also referred toas “the compositions” hereafter) may be prepared from culture solution(it may include fungal cells) obtained by culturing a microorganismcapable of producing a diglycosidase. The microorganisms that may beemployed include those known for their diglycosidases activities, suchas Genus Aspergillus, Genus Penicillium, Genus Rhizopus, GenusRhizomucor, Genus Talaromyces, Genus Mortierella, Genus Cryptococcus,Genus Microbacterium, Genus Corynebacterium, and Genus Actinoplanes.

Examples of more preferred microorganisms include Aspergillus niger (forexample, the IFO4407 strain (available from Institute for Fermentation,Inc., at 2-17-85, Juso-honmachi, Yodogawa-ku, Osaka), IAM 2020 strain),Aspergillus fumigatus (for example, the IAM2046 strain) and Penicilliummulticolor (for example, the IAM7153 strain, available from Institute ofMolecular and Cellular Biosciences, the University of Tokyo at 1-1-1,Yayoi, Bunkyo-ku, Tokyo).

A particularly preferred microorganism is Penicillium multicolorIAM7153. This is particularly preferable because β-galactosidase whichis derived from Penicillium multicolor is designated as a safe enzyme onthe official List of Food Additives and thus a high level of safety ispresumed from compositions obtained from such a safe fungus.

To produce the components of the present invention with theaforementioned microorganisms, any processes and conditions suitable forculturing these microorganisms may be selected. For example, althougheither liquid culture or solid culture may be employed, liquid cultureis the preferred method for culturing the aforementioned microorganisms.The following is an example of liquid culture.

Any culture medium may be used as long as it permits growth ofmicroorganisms producing diglycosidases. Examples include mediacontaining carbon source such as glucose, sucrose, gentiobiose, solublestarch, glycerin, dextrin, molasses, organic acid and the like, nitrogensource such as ammonium sulfate, ammonium carbonate, ammonium phosphate,ammonium acetate, peptone, yeast extract, corn steep liquor, caseinhydrolysate, bran, meat extract and the like, and mineral salts such aspotassium sat, magnesium salt, phosphate, manganese salt, ferrate, zincsalt and the like.

Furthermore, various inducers can be added to the medium to produce andaccumulate diglycosidases. For example, saccharides may be used as theinducers, including such preferred saccharides as gentose (for example,gentose #80 available from Nihon Shokuhin Kako Co., Ltd.), gentiobiose,gentio-oligosaccharide (for example, the gentio-oligosaccharideavailable from Wako Pure Chemical Industries, Ltd.), and galactomannan.The amount of such inducers added is not limited insofar as theproductivity of diglycosidase is increased. Preferably, the amount ofsuch inducers added is in the range of 0.01-10%.

The medium is cultured at the temperatures typically of about 10-50° C.,preferably about 25-30° C. under aerobic conditions for about one tofifteen days, preferably about four to seven days, with the mediumadjusted to a pH of, for example, about 3-8, preferably about 5-6. Themethods of culture that can be employed include shaking culture andaerobic submerged culture in a jar fermenter. However, the foregoingconditions may be altered according to the microorganism or cell to becultured, and are not limited to the foregoing, insofar as conditionssuitable for producing the compositions of the present invention areprovided.

The culture solution or the fungal cells obtained from cultivation for adesired period of time by any of the above-described methods is capableof providing the composition according to the present invention. Forexample, the composition of the present invention may be prepared byfiltration of the culture supernatant followed by, as required,concentration, buffer substitution, sterilization by filtration, andfreeze drying. The composition of the present invention may be either inthe solid state (including the power state) or the liquid state.

Alternatively, the composition of the present invention may be purifiedby a combination of conventional processes, such as centrifugalseparation, purification by ultrafiltration, salting out, and variouschromatographies, such as ion-exchange resin chromatography (see Horio,T., “Basic Experimentation of Proteins and Enzymes”, published byNankodo Co., LTD.)

The composition according to the present invention containsdiglycosidase. In other words, the composition according to the presentinvention has diglycosidase activity. Preferably, the compositionaccording to the present invention has diglycosidase activity of 0.0001units/mg or more in dry weight. Although no upper limit needs to be seton the diglycosidase activity, the maximum value may be, for example, 20units/mg.

As used herein, diglycosidase refers to a sugar-chain hydrolytic enzymecapable of recognizing as a substrate a glycoside, which is composed ofthe non-sugar portion of the glycoside (referred to as an aglyconhereafter) and a branching or straight-chain sugar chain (which is inturn composed of one or more kinds of saccharides), with the non-sugarportion being bonded with the sugar chain via a hydroxyl of the sugarchain. Upon recognizing the substrate in units of disaccharide,diglycosidase cuts the substrate to produce the aglycon. In thisspecification, the diglycosidase activity is quantified by the followingmethod:

Method of Measuring Diglycosidase Activity

In an automatic chemical analyzer (TBA-30R, manufactured by ToshibaCorporation), a sample solution (30 μL) containing a prescribed amountof the composition is mixed with 200 μL of solution in whichp-nitrophenyl (pNP) primeveroside (2 mM) is dissolved in acetate buffer(a pH of 5.5). After the mixture is allowed to react at 40° C. with a22.5 second cycle time for 9.75 minutes, 250 μL of sodium carbonate isadded and the absorbance is then measured at a wave length of 412 nm.Instead of the sample solution, 20 mM acetate buffer (pH 5.5) was usedas a reference for measurement in an identical manner. The amount ofenzyme that increases the absorbance by one under these conditions isreferred to as one unit (AU).

The aforementioned pNP-primeveroside was synthesized by using enzymaticxylosidase (manufactured by Sigma Japan) to cause reaction betweenpNP-glucoside (manufactured by Merck & Co., Inc.) andxylooligosaccharide (manufactured by Wako Pure Chemical Industries,Ltd.) so as to transfer a single residue of xylose to pNP-glucosideforming a β-1,6 bond.

Preferably, the composition of the present invention containsa-L-rhamnosidase as well as diglycosidase. In particular, therhamnosidase activity is preferably 0.0001 units/mg or more in dryweight. Although there is no specific need to set an upper limit on therhamnosidase activity, the maximum value may be, for example, 30units/mg. Alpha-L-rhamnosidase is an enzyme that hydrolyzes thea-L-rhamnopyranoside residue at the non-reducing end of a polysaccharideincluding a-rhamnose. In this specification, the rhamnosidase activityis quantified by the following method:

Method of Measuring Rhamnosidase Activity

In an automatic chemical analyzer (TBA-30R, manufactured by ToshibaCorporation), a sample solution (30 μL) containing a predeterminedamount of the composition is mixed with 200 μL of solution in whichp-nitrophenyl (pNP)-a-rhamnoside (2 mM) (manufactured by Sigma Japan) isdissolved in acetate buffer (a pH of 5.5). After the mixture is allowedto react at 40° C. with a 22.5 second cycle time for 9.75 minutes, 250μL of sodium carbonate is added and the absorbance is then measured at awave length of 412 nm. Instead of the sample solution, 20 mM acetatebuffer (pH 5.5) was used as a reference for measurement in an identicalmanner. The amount of enzyme that increases the absorbance by one underthese conditions is referred to as one unit (AU).

In another aspect, the present invention relates to a method of makingan alcoholic beverage made from grapes with one of the foregoingflavor-improving compositions and is characterized by including anenzymatic process step of causing the flavor-improving composition tooperate (which may be simply referred to as “the enzymatic processstep”).

There is no limitation to the types of alcoholic beverages to which thepresent invention may be applied. The grape-based alcoholic beverageswithin the scope of the present invention include wine, brandy, such ascognac, which is a liquor distilled from wine, and mixed alcoholicbeverages, such as vermouth, port, and liqueur. As used herein, wineincludes a variety of types, such as red, white, rosé, and sparkling.Additionally, as used herein, wine concentrate, which may be laterdiluted or added to other food products, is also considered wine.

Preferably, the enzymatic process step according to the presentinvention is performed simultaneously with part of a standard process ofmanufacturing the grape-based alcoholic beverage. The reason for this isthat since no separate enzymatic process step is required, themanufacturing efficiency is improved.

For example, a standard process of making red wine includes the steps ofcrushing and destemming, fermentation, squeezing, sediment removal, andaging, and any one or more of these steps may be performed under thepresence of the composition of the present invention. Furthermore, astandard process of making white wine includes the steps of crushing anddestemming, squeezing, fermentation, sediment removal, and aging, andany one or more of these steps may be performed under the presence ofthe composition.

By performing one or more of the process of making wine under thepresence of the composition of the invention present as described above,the intended process(es) of the step(s) and the enzymatic process areperformed simultaneously.

In the case of wine (including red, white, and rose), the enzymaticprocess takes place preferably concurrently with the crushing anddestemming, squeezing, fermentation, sediment removal, and/or aging.Preferably, the composition of the present invention is added for theenzymatic process during the fermentation and/or aging steps becauseeach of these steps provides suitable temperature conditions for theenzymatic process and because, in the case of red wine, part of thefruit skin tissue that has been crushed provides conditions suitable forthe operation of the enzyme.

If a step(s) of the standard process is performed under the presence ofthe composition of the present invention as described above, thatcomposition need not to be added at the beginning of the step(s).Rather, the composition may be applied at any suitable time during thatstep. In this way, the duration of the operation of the enzyme isadjustable, thus providing for desired flavor improvement. During theenzymatic process, the processing temperature of the step can beadjusted to achieve good enzyme reaction.

Alternatively, the enzymatic process step according to the presentinvention may be performed as a separate step. If this enzymatic processstep is performed concurrently with another step, it may be necessary toconsider how the addition of the enzyme affects the processingefficiency and other aspects of this concurrent step. However, aseparately performed enzymatic process step will eliminate the need forsuch consideration. This means that favorable temperature and pHconditions can be freely provided, thus enabling efficient enzymaticprocessing.

If performed as a separate step, the enzymatic process step may takeplace before or after any one or more of the steps of the foregoingstandard manufacturing process (for example, the standard processincludes, in red wine making, the steps of crushing and destemming,fermentation, squeezing, sediment removal, and aging, and in white winemaking, the steps of crushing and destemming, squeezing, fermentation,sediment removal, and aging).

Preferably, the added composition should be removed by filtration withbentonite or thermal processing, at a suitable time after the enzymaticprocess step. The sediment removal step of the standard manufacturingprocess may also serve as this step of removing the added composition.

The composition according to the present invention may be directly addedin either a liquid or solid (including power) form to the liquid crushedout of grapes (extract and fruit juice).

The amount of the composition of the present invention used in theenzymatic process step may be decided as desired, depending on the typeof the grape used, the degree of flavor improvement required, thecondition of the crushed grape liquid in the enzymatic process step. Forexample, if the enzymatic process is preformed by adding the enzymebefore or during the fermentation step, the amount of diglycosidase tobe added is decided to provide 0.0026-26000 units of diglycosidase forevery 100 ml of crushed grape liquid obtained from 160 grams of grape. Apreferred amount of diglycosidase used for this purpose is 0.026-2600units and a more preferred amount is 0.26-260 units.

It should be noted that if an insufficient amount of the composition ofthe present invention is used, desired flavor improvement may not beobtained. Conversely, an excessive amount of the composition used maynot only increase the manufacturing cost, but also it may impair theflavor of the beverage due to the effect of and the taste of thecontaminants in the composition.

The temperature range for the operation of the added composition isnormally from 4-40° C., preferably 10-30° C., and more preferably 15-25°C. If the temperature at which the enzyme operates is below theaforementioned normal range, the enzyme in the composition is notallowed to operate fully, such that sufficient flavor improvement is notlikely to occur. If the temperature is above the normal range, theenzyme in the composition becomes more susceptible to deactivation, andthe components in the crushed grape liquid may undergo thermaldenaturation, thus impairing the flavor.

Further, the pH range for the operation of the composition of thepresent invention is normally from 1.5-6.5, preferably 2.0-5.5, and morepreferably 2.5-4.5. If the pH is below or above the aforementionednormal range, the enzyme in the composition is not allowed to operatefully, such that sufficient flavor improvement is not likely to occur,and the components in the crushed grape liquid may undergo thermaldenaturation, thus impairing the flavor.

The following describes the present invention in detail by referring tospecific examples. However, the present invention is not limited tothese examples.

EXAMPLE 1 Preparation of a Flavor-Improving Composition Derived from aMicroorganism

(1-1) Culture of Penicillium multicolor, Strain IAM7153

A culture medium (pH 5.6) containing 2.0% defatted soybean, 3.0%glucose, 0.5% potassium dihydrogenphosphate, 0.4% ammonium sulfate, and0.3% dried yeast was sterilized at 121° C. for 20 minutes. Every 100 mlof the sterilized medium was inoculated with one platinum-loop of thefungus, and was subjected to pre-culture at 27° C. at a shaking rate of140 times per minute. Five days later, 20 liters of the main medium (pH4.9) containing 1.0% Sun Fiber R, 2.0% potassium dihydrogenphosphate,1.0% ammonium sulfate, and 3.13% Myeast P1G, was sterilized in a jarfermenter having a capacity of 30 liters, for 20 minutes at 121° C.while agitating it 150 times per minute. This main culture medium wasthen inoculated with the medium of the foregoing preliminary culture ata final concentration of 1.5% (v/v) and cultured at the airflow rate of0.75 vvm (15 liters/min), an internal pressure of 0.5 kg/cm² (48 kPa)and 27±1° C. for eight days.

(1-2) Preparation of a Flavor-Improving Composition from CultureSolution

Filtration facilitators, Zemlight Super 56M and Fine Flow A (2% each ofthe entire liquid volume), were added to the culture broth to performdiatomaceous earth filtration. The filtered broth was concentrated 20times with an MW 6,000 ultrafilter (UF AIP-2020) and was substitutedwith 20 mM acetate buffer (pH 4.7). The obtained material was sterilizedby filtration and freeze-dried so as to produce the flavor-improvingcomposition.

EXAMPLE 2 Evaluation of the Improvement of the Wine Flavor

(2-1) Preparation of Fruit Juice (Crushed Grape Liquid) from White WineGrapes (the Kelner Variety, Produced in Hokkaido)

Juice from Kelner grapes, a German variety, was prepared as follows:Twelve kilograms of frozen Kelner grapes was first thawed, and thencrushed and destemmed. During this process, 780 mg of potassiumpyrosulfite was added, such that the final content of this additive was100 mg/L (calculated based on the estimated liquid squeeze rate of 65%)The pericarp components were extracted by skin contact at roomtemperature for two hours. Thereafter, this was pressed through nylonmesh cloth, resulting in 7.5 liters of fruit juice. The percentage byspecific gravity of sugar in the fruit juice was measured to give aspecific gravity of 1.0847 and a percentage of sugar inverted from thejuice of 20.18.

(2-2) Alcohol Fermentation, Enzymatic Process, and Analysis ofComponents

To perform alcohol fermentation, an appropriate amount of yeast (thetrade name Uvaferm BC, manufactured by Novozymes Japan, Ltd.) wassuspended in warm water (40° C.) to obtain a 0.1g/ml concentration.After the suspension was left standing for five minutes for activation,15 ml of it was then added to the fruit juice for fermentation.

To produce enzyme reaction, the flavor-improving component of Example 1was added to this fruit juice under the conditions shown in Table 1below. As comparative examples, instead of the flavor-improvingcomposition, a β-glucosidase composition commercially available foraugmenting the aroma of white wine was used to produce enzyme reaction.TABLE 1 Enzyme composition Timing of addition Amount (units) No additive— 0 Flavor-improving At the start of fermentation 7.8 composition At thestart of fermentation 39 At the start of fermentation 195 At the startof fermentation 975 At the start of fermentation 3900 At the start offermentation 7800 After fermentation 7.8 β-glucosidase At the start offermentation 65.6 composition At the start of fermentation 656 At thestart of fermentation 6560 After fermentation 65.6

In the group of samples to which the flavor-improving composition wasadded, the amount of the enzyme composition added is indicated in termsof the diglycosidase activity in the composition added. Likewise, in thegroup of samples to which the β-glucosidase composition was added, theamount of the enzyme composition added is indicated in terms of theβ-glucosidase activity in the composition added. The β-glucosidaseactivity was measured as follows:

Method of Measuring β-Glucosidase Activity

In an automatic chemical analyzer (TBA-30R, manufactured by Toshiba), asample solution (30 μL) containing a prescribed amount of thecomposition is mixed with 200 μL solution in which 2 mM of p-nitrophenyl(pNP)-β-glucoside (manufactured by Sigma Japan) is dissolved in acetatebuffer (a pH of 5.5). After the mixture is allowed to react at 40° C.with a 22.5 second cycle time for 9.75 minutes, 250 μL of sodiumcarbonate is added and the absorbance is then measured at a wave lengthof 412 nm. Instead of the sample solution, 20 mM acetate buffer (pH 5.5)was used as a reference for measurement in an identical manner. Theamount of enzyme that increases the absorbance by one under theseconditions is referred to as one unit (AU).

The addition of the enzyme composition and the enzyme reaction wereeffected by the following two methods: In Method (1), the enzymecomposition was added simultaneously with the yeast to allow alcoholfermentation and enzyme reaction concurrently, whereas in Method (2),after the yeast was added to the fruit juice for alcohol fermentation,the enzyme composition was added for enzyme reaction. More specifically,in the former method or Method (1), after the yeast and the enzymecomposition were added to the fruit juice, this mixture was leftstanding at 20° C. for seven days to effect alcohol fermentation andenzyme reaction. Sampling was conducted on the second, forth, andseventh days (at the end of the fermentation). Centrifugal separationwas performed on each sample at 3000 rpm for 10 minutes and the obtainedsupernatant was subjected to various analyses. The samples were analyzedfor the pH, alcohol content, titratable acidity (with N/10 sodiumhydroxide), sensory evaluation, quantity of terpene componentsresponsible for the aroma with GC/MS equipment.

In the latter method or Method (2), after only the yeast was added, thefruit juice was left standing to ferment at 20° C. for seven days,whereupon the enzyme was added and the fermented juice was additionallyleft standing at 20° C. for one month. Samples were taken uponcompletion of the enzyme reaction. Centrifugal separation was performedon each sample at 3000 rpm for 10 minutes and the obtained supernatantwas subjected to various analyses. In this case, the samples wereanalyzed for the general factores (the pH, alcohol content, andtitratable acidity (with N/10 sodium hydroxide)), sensory evaluation,quantity of terpene components responsible for the aroma with GC/MSequipment.

Table 2 shows the results of the analyses of the samples obtained byMethod (1) (in which alcohol fermentation and enzyme reaction occurconcurrently) for the general components and factors (the pH, alcoholcontent, and titratable acidity (with N/10 sodium hydroxide)). TABLE 2Enzyme Amount titratable acidity pH Alcohol composition added (unit) Day2 Day 4 Day 7 Day 2 Day 4 Day 7 Day 2 Day 4 Day 7 No additives — 10.19.4 9.5 3.9 3.9 4.0 2.5 9.9 12.7 Flavor improving 7.8 9.5 9.4 9.5 3.83.8 3.9 2.2 9.8 13.3 composition 39 9.7 9.3 9.5 3.8 3.8 3.9 2.2 10.313.4 195 9.4 9.2 9.4 3.8 3.8 4.0 2.5 10.5 13.4 975 9.6 9.1 9.7 3.8 3.84.0 2.9 11.3 13.3 3900 9.8 9.7 9.8 3.8 3.8 4.1 3.2 12.5 13.3 7800 9.79.3 10.1 3.9 3.9 4.2 3.6 12.5 12.4 β-glucosidase 65.6 9.2 9.1 9.2 3.93.9 4.0 1.4 9.5 13.2 composition 656 8.7 8.8 9.3 3.9 3.9 4.0 1.4 9.813.2 6560 9.9 9.2 10.5 3.9 3.9 4.0 1.9 11.7 13.5

In the group of samples to which the flavor-improving composition wasadded, the amount of the enzyme composition added is indicated in termsof the diglycosidase activity in the composition added. Likewise, in thegroup of samples to which the β-glucosidase composition was added, theamount of the enzyme composition added is indicated in terms of theβ-glucosidase activity in the composition added.

The results showed no significant change in the values for these factorscaused by the addition of the flavor-improving composition. In additionto these factors, the samples were analyzed for the polyphenol content,specific gravity, and color tone. The results showed no significantchange in these factors due to the addition of the flavor-improvingcomposition, either (no data provided). The foregoing results show thataddition of the flavor-improving composition has little effect on thegeneral components (factors), and that it is capable of enzymaticprocessing without affecting the overall quality of the product (whichis evaluated on the basis of the above factors.

Table 3 shows the results of sensory evaluation of the samples producedby Method (1) upon the completion of the fermentation (on the seventhday). Eight panelists were assembled to grade the strength of theterpene aroma, which is regarded as an important characteristic inmuscat wine and wine made from German grape varieties, on the scale offive (5: strong, 4: fairly strong, 3: average, 2: fairly weak, 1: weak).For each sample, the panelists' grades were totaled and averaged. TABLE3 Enzyme Amount added Panelists composition (units) A B C D E F G HTotal Average No additives — 3 2 4 2 2 3 3 3 22 2.8 Flavor-improving 7.84 4 3 3 4 4 5 2 29 3.6 composition 39 3 3 2 3 5 4 4 3 27 3.4 195 2 4 4 43 3 2 1 23 2.9 β-glucosidase 65.6 3 2 3 4 3 4 4 3 26 3.3 composition 6562 1 3 3 2 4 4 3 22 2.8 6560 2 3 2 3 1 3 2 3 19 2.4

In the group of samples to which the flavor-improving composition wasadded, the amount of the enzyme composition added is indicated in termsof the diglycosidase activity in the composition added. Likewise, in thegroup of samples to which the β-glucosidase composition was added, theamount of the enzyme composition added is indicated in terms of theβ-glucosidase activity in the composition added.

Table 3 shows that the addition of the flavor-improving compositionclearly augments the terpene aroma.

Table 4 shows the results of GC/MS analyses for the componentsresponsible for the terpene aroma. TABLE 4 Enzyme Amount added Linaloola-terpineol Citronellol composistion (unit) Day 2 Day 4 Day 7 Day 2 Day4 Day 7 Day 2 Day 4 Day 7 No additives 0 41.6 41.0 48.6 N.D N.D. 14.7N.D. 8.2 34.8 Flavor improving 7.8 43.6 44.4 82.8 3.2 N.D. 9.0 N.D. N.D.12.9 composition 39 42.2 67.4 80.7 N.D. N.D. 5.9 N.D. N.D. 14.0 195 45.367.9 146.2 N.D. N.D. 12.8 N.D. N.D. 13.9 975 56.5 145.2 233.3 N.D. 12.730.3 N.D. N.D. 13.6 3900 111.7 226.3 297.6 14.7 33.8 60.3 N.D. N.D. 18.07800 170.0 333.1 299.3 27.2 59.4 88.6 N.D. 21.1 34.8 β-glucosidase 65.639.4 51.6 50.9 N.D. N.D. N.D. N.D. N.D N.D. composition 656 43.2 53.256.7 N.D. N.D. 4.8 N.D. N.D 13.9 6560 46.4 62.9 118.0 N.D. N.D. 6.8 N.D.N.D 17.7 German wine — — 6.1 — — 41.9 — — N.D. Enzyme Nerol Geranioltotal composistion Day 2 Day 4 Day 7 Day 2 Day 4 Day 7 Day 2 Day 4 Day 7No additives N.D. N.D. 19.5 N.D N.D. 43.9 41.6 49.2 161.5 Flavorimproving N.D. N.D. 46.4 N.D. N.D. N.D. 46.8 44.4 151.1 composition N.D.N.D. 32.8 N.D. N.D. N.D. 42.2 67.4 133.4 10.4 42.1 37.6 13.5 N.D. N.D.69.2 110.0 210.5 11.7 57.2 24.4 14.0 N.D. N.D. 82.2 215.1 301.5 N.D.31.9 N.D. N.D. N.D. N.D. 126.4 332.0 375.9 N.D. N.D. N.D. N.D. N.D. N.D.197.3 413.5 442.7 β-glucosidase N.D. 19.5 10.7 12.6 N.D. N.D. 52.0 71.261.6 composition N.D. 23.2 21.4 N.D. N.D. 16.1 43.2 76.2 113.0 N.D. 60.526.0 36.0 N.D. 14.4 83.0 123.4 182.9 German wine — — N.D. — — N.D. — —47.9

In the group of samples to which the flavor-improving composition wasadded, the amount of the enzyme composition added is indicated in termsof the diglycosidase activity in the composition added. Likewise, in thegroup of samples to which the β-glucosidase composition was added, theamount of the enzyme composition added is indicated in terms of theβ-glucosidase activity in the composition added.

As shown in Table 4, although the amount of nerol or geraniol did notincrease in accordance with the amount of the flavor-improvingcomposition added (probably due to the fact that the experiment was on asmall-scale, such that these components were affected, for example,oxidized during the enzyme reaction), the amounts of the other aromacomponents did increase in accordance with the amount of theflavor-improving composition added. It is also observed that the contentof each aroma component changed generally in keeping with the number ofdays from the addition. It is additionally shown that in terms of theamount added, the flavor-improving composition more effectively augmentsthe aroma components than the commercially available β-glucosidasecomposition. The bottom row of Table 4 shows the measurements of thecomponents responsible for the aroma in a commercially available Germanwine.

Tables 5 and 6 show, respectively, the results of sensory evaluation andGC/MS analyses of the samples produced by Method (2), in which thefermentation occurred after the enzyme reaction. In the sensoryevaluation, the panelists chose the one that they felt had a strongerterpene aroma between the sample obtained by using the flavor-improvingcomposition and the sample obtained by using the commercially availableβ-glucosidase composition. TABLE 5 Chosen as Enzyme Amount morecomposition added aromatic Comments Flavor- 7.8 By 6 A fresh fragrance.A lightly improving panelists flowery fragrance. Balmy. composition Amellow fragrance. Grapy. Slightly pungent. Refreshing β-glucosidase 65.6By 1 A subdued fragrance. Moderately composition panelist strong greenfragrance. A sour- sweet smell. Tastes like a medicine. Full-bodied.

In the group of samples to which the flavor-improving composition wasadded, the amount of the enzyme composition added is indicated in termsof the diglycosidase activity in the composition added. Likewise, in thegroup of samples to which the β-glucosidase composition was added, theamount of the enzyme composition added is indicated in terms of theβ-glucosidase activity in the composition added.

Table 5 confirms that the addition of the flavor-improving compositionclearly augments the terpene aroma of wine more than the addition of thecommercially available β-glucosidase composition. Since these samplegroups give different aromatic impressions, presumably, theflavor-improving composition is capable of providing a novel aromaaugmenting effect distinct from that provided by the exsitingβ-glucosidase composition. TABLE 6 Enzyme Amount added composition(units) linalool a-terpineol citronellol nerol geraniol Total Noadditives 0 72.5 13.9 9.4 3.6 N.D. 99.4 Flavor-improving 7.8 132.7 24.7N.D. 37.6 N.D. 195.0 composition β-glucosidase 65.6 94.3 15.0 N.D. N.D.N.D. 109.3 composition

In the group of samples to which the flavor-improving composition wasadded, the amount of the enzyme composition added is indicated in termsof the diglycosidase activity in the composition added. Likewise, in thegroup of samples to which the β-glucosidase composition was added, theamount of the enzyme composition added is indicated in terms of theβ-glucosidase activity in the composition added.

Table 6 shows a higher content of each component responsible for thearoma in the samples produced with the flavor-improving composition,substantiating the results of the foregoing sensory evaluation.Moreover, nerol was detected in the samples produced with theflavor-improving composition, indicating flavor improvement that isqualitatively different from that achieved by the commercially availableβ-glucosidase.

EXAMPLE 3 Determination of the Amounts of the Flavor-ImprovingComposition to be Added and Comparison with Other Enzyme Compositions

In order to further determine the relationship between the amount of theflavor-improving composition added and the effect of augmenting thecomponents responsible for wine aroma, additional batches of white winewere prepared by narrowing the amounts of the enzymes added. In thisexperiment, samples in which the flavor-improving composition was usedfor enzyme processing were compared with those in which a commerciallyavailable β-glucosidase composition was used for enzyme processing.Table 7 shows the diglycosidase (DGL) activity, the rhamnosidase (Rhm)activity, and the β-glucosidase activities (Glc) of the flavor-improvingcomposition and the β-glucosidase composition used. TABLE 7 Enzymeactivity (unit/g) Enzyme diglycosidase rhamnosidase glucosidasecomposition activity(*) activity(**) activity(***) Flavor-improving 260038532 1920 composition β-glucosidase 2.2 15.6 2187 composition(*)Measured with pNP-β-primeveroside as the substrate.(**)Measured with pNP-a-rhamnoside as the substrate.(***)Measured with pNP-β-glucopyranoside as the substrate.

Following a process identical to the one in Example 2, Kelner fruitjuice was prepared using 6 kg of Kelner grapes as the starting material.The specific gravity of the fruit juice was 1.0794 and the percentage offruit sugar from the juice was 18.83.

After the fruit juice was dispensed, yeast (the trade name Uvaferm BC,manufactured by Novozymes Japan, Ltd.) and then an enzyme compositionwere added to each dispensation. A suitable amount of water was added tothe yeast so as to obtain a 0.1 g/ml concentration, and the watersolution was left standing at 40° C. for five minutes to permitactivation. Fifteen milliliters of it was then added to the fruit juice.A predetermined amount of each enzyme composition was dissolved in waterand added to the fruit juice in the form of aqueous solution. Table 8below shows the amount of each enzyme composition and the charge of eachsolution containing the enzyme composition. TABLE 8 Amount of additionEnzyme compsotion (Units/charge) Charge (ml) No addition 0 300Flavor-improvement composition 3.9 300 7.8 300 23.4 300 β-glucosidasecomposition 2.0 200

In the group of samples to which the flavor-improving composition wasadded, the amount of the enzyme composition added is indicated in termsof the diglycosidase activity in the composition added. Likewise, in thegroup of samples to which the β-glucosidase composition was added, theamount of the enzyme composition added is indicated in terms of theβ-glucosidase activity in the composition added.

After the yeast and the enzyme composition were added, the fruit juicewas left standing at 20° C. to effect alcohol fermentation and enzymereaction. Upon completion of the fermentation (on the sixth day), 20 mlsamples were taken. Each sample was centrifuged at 3000 rpm for 10minutes. The resultant supernatant was analyzed for the general factors(the titratable acidity (with N/10 sodium hydroxide), pH, and alcoholcontent) and the components responsible for the aroma, and alsosubjected to sensory evaluation.

A thermo desorption system and an Agilent GC/MSD system (manufactured byGRESTEL K.K.) were used for the analysis of the amounts of the aromacomponents. A column (HP-INNO Wax Polyethylene Glycol (30.0m×250 μm×0.25μm)) was used for this analysis with helium as the mobile phase and at aflow rate of 1. Oml/min. The evaporation temperature was held at 40° C.for five minutes, raised at the rate of 5° C./min to 240° C., and heldat this point for 15 minutes. The split ratio was 1:50. Additionally,2.0g of NaCl and an internal control (4-Nonanol, 5 μg) were added to 10ml of each sample, which was then stirred with a stirrer (Twister,manufactured by GERSTEL K.K.) for 30 minutes before conducting theanalysis. The following are the aroma components (monoterpene alcohols)for this measurement and the m/z values used for the quantification.

-   -   Internal control: 4-Nonanol (m/z=101)    -   Linanol (m/z=93)    -   a-terpineol (m/z=121)    -   Citronellol (m/z=123)    -   Nerol (m/z=93)    -   Geraniol (m/z=123)

As the factor for quantification, approximately 1000 ppb of monoterpenealcohol was added to 0.5% malic acid solution so as to perform analysisin the same manner as the samples.

Table 9 shows the results of the sample analyses performed at thecompletion of fermentation for the titratable acidity (with N/10 sodiumhydroxide solution), pH, and alcohol content. TABLE 9 Enzyme AmountTitratable acidity Alcohol composition added (units) Timing of additionN/10 NaOH (ml) pH (w/w %) No additives 0 9.2 3.61 11.35 Flavor-improving3.9 Simultaneously with fermentation 9 3.58 11.35 composition 7.8Simultaneously with fermentation 9.1 3.58 11.35 23.4 Simultaneously withfermentation 9.2 3.6 11.35 β-glucosidase 2.0 Simultaneously withfermentation 8.7 3.56 11.25 composition

In the group of samples to which the flavor-improving composition wasadded, the amount of the enzyme composition added is indicated in termsof the diglycosidase activity in the composition added. Likewise, in thegroup of samples to which the β-glucosidase composition was added, theamount of the enzyme composition added is indicated in terms of theβ-glucosidase activity in the composition added.

The results showed no significant change in the values for thesecomponents among the samples to which the flavor-improving compositionwas added. Neither did varying the added amounts result in anysignificant changes in the values for these components. The foregoingresults confirm that the flavor-improving composition is capable ofenzymatic processing while hardly affecting the general componentsselected for measurement, that is, the flavor-improving composition iscapable of enzymatic processing without affecting the overall quality ofthe product (which is evaluated on the basis of these general componentsor factors.

Table 10 shows the results of analyses for the amounts of the componentsresponsible for the aroma. TABLE 10 Enzyme Amount Linalool a-terpineolCitronellol composistion added (unit) Timing of addition Day 0 Day 6 Day0 Day 6 Day 0 Day 6 No additives 0 30.95 35.75 N.D N.D. N.D. 22.58Flavor improving 3.9 Simultaneously — 40.87 — 4.5 — 15.99 compositionwith fermentation 7.8 Simultaneously — 41.87 — 4 — 16.32 withfermentation 23.4 Simultaneously — 52.43 — 4.72 — 19.79 withfermentation β-glucosidase 2.0 Simultaneously — 35.46 — 3.09 — 14.28composition with fermentation Enzyme Nerol Geraniol Total composistionDay 0 Day 6 Day 0 Day 6 Day 0 Day 6 No additives 6.25 N.D. 9.71 20.5346.91 78.86 Flavor improving — N.D. — 21.62 — 82.98 composition — N.D. —20.95 — 82.74 — 14.32 — 29.74 — 121 — 7.11 — N.D. — 59.94 β-glucosidasecomposition

In the group of samples to which the flavor-improving composition wasadded, the amount of the enzyme composition added is indicated in termsof the diglycosidase activity in the composition added. Likewise, in thegroup of samples to which the β-glucosidase composition was added, theamount of the enzyme composition added is indicated in terms of theβ-glucosidase activity in the composition added.

As shown in Table 10, among the samples to which the flavor-improvingcomposition had been added, the content of each component responsiblefor aroma increased generally in proportion to the amount of thecomposition added.

Ten panelists were assembled to conduct an sensory test on samplesprepared upon completion of the fermentation. The panelists graded thestrength of the terpene aroma (including its strength on the palate) onthe scale of five (5: strong, 4: fairly strong, 3: average, 2: fairlyweak, 1: weak) for each sample, and the grades were totaled andaveraged. Table 11 shows the results of the sensory test. TABLE 11Enzyme Amount added Panelists Composition (units) Timing of addition A BC D E F G H I J Total Average No additives 0 2 3 2 3 3 2 3 3 4 2 27 2.7Flavor-improving 3.9 Simultaneously 2 2 4 2 2 1 3 1 3 3 23 2.3Composition with fermentation 7.8 Simultaneously 4 2 4 5 4 2 4 1 2 2 303.0 with fermentation 23.4 Simultaneously 5 4 3 4 4 3 4 1 4 3 35 3.5with fermentation β-glucosidase 2.0 Simultaneously 3 3 2 3 2 1 4 3 4 126 2.6 composition with fermentation

In the group of samples to which the flavor-improving composition wasadded, the amount of the enzyme composition added is indicated in termsof the diglycosidase activity in the composition added. Likewise, in thegroup of samples to which the β-glucosidase composition was added, theamount of the enzyme composition added is indicated in terms of theβ-glucosidase activity in the composition added.

The results of the evaluation indicate that the group of samples towhich the flavor-improving composition was added provides approximatelythe same level of terpene aroma as the control (no enzyme added) or astronger level than the control. The sample to which 3.9 units was addedreceived lower grades than the control, showing such a small additiondoes not sufficiently augment the components responsible for the aroma.Meanwhile, the sample that received 7.8 units was given significantlyhigher grades, indicating this amount provides sufficient augmentationof the aroma components. It is also confirmed that the sample to whicheven more units, i.e., 39 units, was added further augmented thecomponents responsible for the aroma. These results indicate that addingat least 7.8 units of the flavor-improving composition augments thecomponents responsible for the aroma.

EXAMPLE 4 Evaluation of the Improvement of the Flavor of Red Wine

The improvement of the flavor of red wine was evaluated by the followingprocedure: The flavor-improving component of Example 1 was added tocommercially available wine (Bordeaux Superieur, imported by Kokubu &Co., Ltd.) under the conditions in Table 12 below so as to effectenzymatic operation at 20° C. for nine days. Thereupon, each sample wascentrifuged at 3000-rpm for 10 minutes and the obtained supernatant wassubjected to analysis for the components responsible for the aroma. Aβ-glucosidase composition commercially available for augmenting thearoma of white wine was used in comparative examples. TABLE 12 No.Amount of enzyme added to every 30 ml of red wine 1 30 mg offlavor-improving composition &   78 units 0.1 ml of water added: 2 0.1ml of solution containing 30 mg of  7.8 units flavor-improvingcomposition per ml: 3 0.1 ml of solution containing 3 mg of 0.78 unitsflavor-improving composition per ml: 5 0.1 ml of solution containing 30mg of 65.3 units β-glucosidase composition per ml: 7 No enzyme added but0.1 ml of water added: 0

A headspace (HP7694 Headspace Sampler, manufactured by AlilentTechnologies Inc.)-GC/MSD (HP5973 GC/HP6890 MSD, manufactured by AlilentTechnologies Inc.) system was used for the analysis of the amounts ofthe components responsible for the aroma. The column used for thisanalysis was an HP-WAX (60 m×250 μm×0.2 5 μm) with helium as the mobilephase and at a flow rate of 1.0 ml/min. After a sample placed in a vialfor analysis was heated at 85° C. for 20 minutes, the gas was sampledand injected into the GC/MS at a split ratio of 25:1. After beinginitially maintained at 35° C. for 5 minutes, the oven was heated to100° C. at the rate of 5° C./min and further heated to 230° C. at therate of 10° C./min, where it was held for 10 minutes.

In the group of samples to which the flavor-improving composition wasadded, the amount of the enzyme composition added is indicated in termsof the diglycosidase activity in the composition added. Likewise, in thegroup of samples to which the β-glucosidase composition was added, theamount of the enzyme composition added is indicated in terms of theβ-glucosidase activity in the composition added.

Table 13 shows the results of the analysis. TABLE 13 Amount of additiveAmount of additive Peak diglycosidase units β-glucosidase units No Noadditives 78 7.8 0.78 65.3 Name of compound 1 645813 1032273 736483522471 769260 Ethyl propionate 1.0 1.6 1.1 0.8 1.2 2 2297101 1438803212024229 4407696 2393265 Ethyl caprylate 1.0 6.3 5.2 1.9 1.0 3 4943741066060 702584 539129 505635 Benzaldehyde 1.0 2.2 1.4 1.1 1.0 4 N.D.213325 300307 N.D. 137005 Butane-4-oide — 8 8 — 8 5 N.D. 187661 83903N.D. N.D. Methyl salicylate — 8 8 — — 6 118608 296007 195854 60338 87206Benzylalcohol 1.0 2.5 1.7 0.5 0.7

In the group of samples to which the flavor-improving composition wasadded, the amount of the enzyme composition added is indicated in termsof the diglycosidase activity in the composition added. Likewise, in thegroup of samples to which the β-glucosidase composition was added, theamount of the enzyme composition added is indicated in terms of theβ-glucosidase activity in the composition added.

In Table 13, the samples to which no enzyme was added were processed inthe same manner (left standing at 20° C. for nine days) as those towhich the flavor-improving composition was added. Table 13 lists onlythose components that exhibit different results as compared with thosewith no additives (i.e., the controls). For each peak, the upper valuerepresents the peak area, whereas the lower value represents its arearatio to the value for the sample with no additives. It is shown thatthe amounts of such components responsible for the aroma asbenzaldehyde, methyl salicylate, and benzyl alcohol (known to exist inthe form of glycosides as precursors) markedly increased through theoperation of the flavor-improving composition. Additionally, esters,such as ethyl propionate and ethyl caprylate, are shown to haveincreased in amount. Meanwhile, although the amounts of some componentsare shown to have increased in the comparative example, to which theβ-glucosidase composition was added, the increases were modest.Moreover, the operation of the flavor-improving composition imparted asweet, flowery aroma to the wine, substantiating the improvement of thearoma (no data provided).

The results of the foregoing examples confirm that the operation of theflavor-improving composition of the present invention in a winemakingprocess is an effective means to improve the components in wineresponsible for the aroma. Since the flavor-improving compositionaccording to the present invention acts on components in grape juice,from which wine is made, to bring out the aroma components, thisflavor-improving composition is effective in augmenting the aromacomponents not only in white and red wine (used as examples above) butalso in other types of wine, such as rose, as well as other grape-basedalcoholic beverages.

The following is disclosed:

-   (11) A composition as set forth in any one of claims 1-4, wherein    the rhamnosidase activity is 0.0001 units/mg or more in dry weight.-   (21) A method of manufacturing a grape-based alcoholic beverage,    including an enzymatic process step of causing diglycosidase to    operate.-   (22) A method of manufacturing as set forth in (21), wherein the    enzymatic process step includes addition of diglycosidase in part of    the process of manufacturing the grape-based alcoholic beverage.-   (23) A method of manufacturing as set forth in (22), wherein the    part of the process of manufacturing includes one or more steps    selected from the group consisting of squeezing, fermentation,    sediment removal, and aging.-   (24) A method of manufacturing a grape-based alcoholic beverage,    characterized by adding diglycosidase in a fermentation step and/or    an aging step.-   (25) A method of manufacturing as set forth in any one of (21) to    (24), wherein the diglycosidase is derived from a microorganism.-   (26) A method of manufacturing as set forth in any one of (21) to    (24), wherein the diglycosidase is derived from Penicillium    multicolor.-   (27) A method of manufacturing as set forth in any one of (21) to    (24), wherein the diglycosidase is derived from Aspergillus    fumigatus.-   (28) A method of manufacturing a grape-based alcoholic beverage as    set forth in any one of (21) to (27), wherein the grape-based    alcoholic beverage is wine.-   (29) A grape-based alcoholic beverage manufactured by any one of the    methods set forth in (21) to (28).-   (30) A grape-based alcoholic beverage the flavor of which has been    improved by the operation of diglycosidase.-   (31) Wine whose flavor has been improved by the operation of    diglycosidase.-   (41) A method of manufacturing a grape-based alcoholic beverage,    including an enzymatic process step of causing diglycosidase and    rhamnosidase to operate.-   (42) A method of manufacturing as set forth in (41), wherein the    enzymatic process step includes addition of diglycosidase and    rhamnosidase in part of the process of manufacturing the grape-based    alcoholic beverage.-   (43) A method of manufacturing as set forth in (42), wherein the    part of the process of manufacturing includes one or more steps    selected from the group consisting of squeezing, fermentation,    sediment removal, and aging.-   (44) A method of manufacturing a grape-based alcoholic beverage,    characterized by adding diglycosidase and rhamnosidase in a    fermentation step and/or an aging step.-   (45) A method of manufacturing as set forth in any one of (41) to    (44), wherein the diglycosidase is derived from a microorganism.-   (46) A method of manufacturing as set forth in any one of (41) to    (44), wherein the diglycosidase and rhamnosidase are derived from    Penicillium multicolor.-   (47) A method of manufacturing as set forth in any one of (41) to    (44), wherein the diglycosidase and rhamnosidase are derived from    Aspergillus fumigatus.-   (48) A method of manufacturing a grape-based alcoholic beverage as    set forth in any one of (41) to (47), wherein the grape-based    alcoholic beverage is wine.-   (49) A grape-based alcoholic beverage manufactured by any one of the    methods set forth in any one of (41) to (48).-   (50) A grape-based alcoholic beverage the flavor of which has been    improved by the operation of diglycosidase and rhamnosidase.-   (51) Wine whose flavor has been improved by the operation of    diglycosidase and rhamnosidase.

INDUSTRIAL APPLICABILITY

A grape-based alcoholic beverage with improved flavor is provided byaugmentation of the components responsible for its aroma as a result ofapplication of the present invention. More particularly, the presentinvention is capable of providing a grape-based alcoholic beverage withbetter flavor than alcoholic beverages whose flavors are improved withconventional, commercially available β-glucosidase compositions.

Moreover, by adjusting the usage of a composition according to thepresent invention, the degree of augmentation of the componentsresponsible for the aroma may also be adjusted, making it possible toprovide grape-based alcoholic beverages having a variety of aromaticbalances.

Furthermore, any of the compositions according to the present inventionmay be added to operate before or during the fermentation step. Thismeans that there are fewer limits on the timing of adding thesecompositions, thus providing advantages in manufacturing. If addedbefore or during the fermentation step, the compositions of the presentinvention may be removed simultaneously with the yeast, thus simplifyingthe manufacturing process and reducing the manufacturing costs.

1. A composition for improving the flavor of an alcoholic beverage madefrom grapes, the composition containing a culture of a strain belongingto Genus Aspergillus, Genus Penicillium, Genus Rhizopus, GenusRhizomucor, Genus Talaromyces, Genus Mortierella, Genus Cryptococcus,Genus Microbacterium, Genus Corynebacterium, or Genus Actinoplanes, andbeing capable of producing diglycosidase.
 2. A composition according toclaim 1, wherein the strain belongs to Aspergillus niger, Aspergillusfumigatus, or Penicillium multicolor.
 3. A composition according toclaim 1, wherein the strain is Aspergillus niger IFO4407, Aspergillusniger IAM2020, Aspergillus fumigatus IAM2046, or Penicillium multicolorIAM7153.
 4. A composition including an extracellular enzyme produced byPenicillium multicolor IAM7153.
 5. A composition according to claim 1,wherein the diglycosidase activity is 0.0001 units/mg or more in dryweight.
 6. A method of manufacturing a grape-based alcoholic beverage,including an enzymatic process step of causing the flavor-improvingcomposition according to claim 1 to operate.
 7. A method ofmanufacturing according to claim 6, wherein the enzymatic process stepincludes addition of the flavor-improving composition in part of theprocess of manufacturing the grape-based alcoholic beverage.
 8. A methodof manufacturing according to claim 7, wherein said part of the processof manufacturing includes one or more steps selected from the groupconsisting of crushing and destemming, squeezing, fermentation, sedimentremoval, and aging.
 9. A method of manufacturing a grape-based alcoholicbeverage, characterized by adding the composition according to claim 1in the fermentation and/or aging step.
 10. A method of manufacturing agrape-based alcoholic beverage according to claim 6, wherein thegrape-based alcoholic beverage is wine.
 11. A grape-based alcoholicbeverage manufactured by the method according to claim 6.